Cellular adaptation to low oxygen (hypoxia) is an important biological problem not only in relation to pathological conditions such as cancer and ischemic diseases, but also in normal fetal development and in cell differentiation. Our studies have shown that chondrocytes are an excellent model to learn how cells adapt to, and differentiate in a low oxygen environment. The fetal growth plate is a unique mesenchymal tissue since it is avascular, though it requires the angiogenic switch in order to be replaced by bone. We have recently demonstrated that the fetal growth plate has an out-in gradient of oxygenation with a central, hypoxic region. Moreover, we have discovered that the transcription factor Hypoxia-inducible factor-1a (Hif-1a), which is a major mediator of the cellular adaptation to hypoxia, controls critical steps of endochondral bone development. Our work thus far, in fact, suggests that the actions of Hif-1a are central to the regulation of survival and differentiation of chondrocytes;this renewal, will endeavor to understand these vital roles of Hif-1a. Chondrocytes lacking Hif-1a undergo massive cell death, particularly in the center of the developing growth plate. Hif-1a is thus a survival factor, at least for cells of mesenchymal origin such as chondrocytes. Notably, in the fetal growth plate this "central cell death phenotype" is mimicked by genetic ablation of Vascular Endothelial Growth Factor A (VEGF), a direct downstream target of Hif-1a. To start dissecting out the molecular mechanisms downstream of Hif-1a as a survival factor, in Aim I of this grant proposal, we will investigate whether VEGF is able to rescue, at least in part, the loss of cell viability observed in fetal growth plate deficient in Hif-1a, and we will thus gain further insights into the role of VEGF as one of the mediators of the survival function of Hif-1a in the fetal growth plate. Detailed analysis of an in vivo loss-of-function model of Hif-1a in cartilage has provided strong evidence that this transcription factor has also non-redundant functions in controlling differentiation of mesenchymal cells into chondrocytes, as lack of Hif-1a in limb bud mesenchyme considerably delays the formation of the cartilaginous primordia. In addition, preliminary data have shown that hypoxia and Hif-1a regulate terminal stages of chondrocyte differentiation. Collectively, these findings suggest that low oxygen tension, far from being detrimental, is 'required'for formation of cartilage, by up regulating Hif-1a transcriptional activity. In Aim II, we will study the essential role of Hif-1a in chondrocyte differentiation, and we will test the hypothesis that hypoxia and Hif-1a regulate expression of prolyl-4-hydroxylase II, which controls post-translational hydroxylation of collagens in chondrocytes. PUBLIC HEALTH RELEVANCE. If successful this proposal will significantly advance our knowledge of the molecular mechanisms underlying the cellular adaptation to hypoxia and the differentiation process of mesenchymal cells into chondrocytes. Thus, its relevance is high for both organogenesis and mesenchymal stem cell biology.